High Throughput Screening for nAChRs: Cell Lines and Assay Development

描述(由申请人提供)：这项对NIH/NIDA RFA-DA-11-007高度响应的研究项目的目标是开发一种高通量筛选(HTS)试验，用于筛选和鉴定包含a3、a5、a6和/或b4亚单位的神经元烟碱型乙酰胆碱受体(NAChRs)的选择性配体。NAChRs是一种配体门控离子通道，参与多种生理功能、病理过程和药理作用。它们已被证明可以调节尼古丁的成瘾效应，尼古丁是全球超过10亿人吸烟或使用其他烟草产品的主要原因。神经元nAChRs由形成五聚体阳离子通道的a和b亚基组成。已在脊椎动物神经组织中鉴定出9个a亚基(a2-a10)和3个b亚基(b2-b4)。虽然A7亚基形成功能性的同源受体，但哺乳动物大脑中的大多数nAChR是包含两个或更多亚基的异构体受体。多年来，a4b2 nAChR亚型(由a4、b2和其他亚基组成)一直是研究尼古丁成瘾和开发戒烟疗法的主要焦点，许多证据支持它们的参与；但自2007年以来，全基因组关联研究揭示了染色体15q24-25.1上人类a5-a3-b4基因簇的标记与吸烟和吸烟相关疾病的显著关联。在这些标记中，最引人注目的变异是rs16969968，它在a5亚基的第398位(D398N)将氨基酸从天冬氨酸改变为天冬酰胺。这一和最近的发现表明，除了a4和b2亚基之外，a3、a5、a6和b4亚基在吸烟和吸烟相关疾病中发挥着重要作用。鉴于这一新的信息，开发对含有a3、a5、a6和/或b4亚单位的受体具有选择性的新的配体被认为是重要的。因此，迫切需要一种HTS分析方法，能够可靠地在现有或新的化合物文库中识别选择性配体。我们提出了两个具体的目标来实现这一目标：目标1：建立高功能的稳定表达含有a3、a5、a6和/或b4亚单位的人nAChRs的细胞系。目的2：建立一种基于IonFlux自动化高通量膜片钳系统的功能性HTS检测方法。 与公共健康相关：在美国，吸烟是可预防疾病和过早死亡的主要原因。尼古丁受体的选择性配体可用于研究尼古丁成瘾和开发新的戒烟疗法。本项目的目标是开发高通量的筛选方法，以加速选择性配体的发展。

DESCRIPTION (provided by applicant): The goal of this research project, which is highly responsive to NIH/NIDA RFA-DA-11-007, is to develop a high throughput screening (HTS) assay for screening and identifying selective ligands for neuronal nicotinic acetylcholine receptors (nAChRs) containing a3, a5, a6 and/or b4 subunits. nAChRs are ligand-gated ion channels that are implicated in a wide range of physiological functions, pathological processes and pharmacological effects. They have been shown to mediate the addictive effects of nicotine, which are the main reason why more than a billion people worldwide smoke cigarettes or use other tobacco products. Neuronal nAChRs are composed of a and b subunits that form pentameric cation channels. Nine a subunits (a2 - a10) and three b subunits (b2 - b4) have been identified in vertebrate neuronal tissues. Although the a7 subunit forms functional homomeric receptors, most nAChRs in mammalian brain are heteromeric receptors that contain two or more subunits. For many years, a4b2 nAChR subtypes (composed of a4, b2 and perhaps other subunits) have been the primary focus for studying nicotine addiction and developing smoking cessation therapeutics, and much evidence supports their involvement; but since 2007 genome-wide association studies have revealed significant associations of markers in the human a5-a3-b4 gene cluster on chromosome 15q24-25.1 with smoking and smoking related diseases. Among these markers, the most compelling variant is rs16969968, which changes an amino acid from aspartate to asparagine at position 398 (D398N) in the a5 subunit. This and more recent discoveries suggest that in addition to a4 and b2 subunits, a3, a5, a6 and b4 subunits play important roles in smoking and smoking related diseases. In light of this new information, it is considered important to develop new ligands selective for receptors containing a3, a5, a6 and/or b4 subunits. Hence, the crucial need for a HTS assay that can reliably identify selective ligands in existing or new libraries of compounds. We propose two Specific Aims to accomplish this goal: Aim 1: To establish highly functional stably transfected cell lines that express human nAChRs containing a3, a5, a6 and/or b4 subunits. Aim 2: To develop a functional HTS assay using IonFlux automated, high throughput patch clamp system. PUBLIC HEALTH RELEVANCE: Cigarette smoking is the leading cause of preventable disease and premature death in the United States. Selective ligands of nicotinic receptors can be used in studying nicotine addiction and for developing new smoking cessation therapeutics. The goal of this project is to develop high throughput screening assay that will accelerate the development of selective ligands.